Studies On Production Of L-Cysteine Synthetase And Enzymatic Characteristcs Induced By DL-2-amino-Δ~2-thiazoline-4-carboxylic Acid In Pseudomonas Sp. F12

L-Cysteine is the only amino acid containing sulfydryl in the20kinds of amino acid composed of protein. Because of the strongly polar sulfydryl, it possesses special reaction characteristcs. L-cysteine is widely used in pharmaceuticals, foods and comestic industry and new applications are continuously being developed. Microbial production of L-cysteine synthetase (CS) induced by DL-2-amino-Δ2-thiazoline-4-carboxylic acid is a hotspot of research and development today.Various kinds of amino acids are produced by hydrolysis in which the microbial enzymes are induced by cells growing on a sort of substrate as the sole nitrogen source, such as hydantoins and its derivatives, pyrimidine base, and it is the case of microbial production of L-cysteine from DL-ATC. A series of studies have been conducted concerning cloning, expression, enzymatic characteristcs of CS induced by DL-ATC in microbial. In the present study, a strain named Pseudomonas sp. F12was screened out, and two-stage culture for CS production were performed based on the principles of metabolic regulation. In addition, enzymatic characteristcs of CS were preliminary studied. Followings are the main results:A stain, named Pseudomonas sp. F12, was obtained by screening27samples from different areas by cultivating on a minimal medium, in which DL-ATC was the sole nitrogen source. It can grow by utilizing DL-ATC as the sole nitrogen and carbon source, and ammonium was released to the medium as DL-ATC consumed. It also showed enzyme activities after adding carbon or nitrogen source to the minimal medium (which was used), indicating that DL-ATC can induced CS both as carbon source and nitrogen source. Enzyme activities of cells growing on minimal medium was much higher than that growing on rich medium, which suggested that enzyme expression probably transcriptionally was tightly regulated.The effect of glucose on CS production and the rule of CS production were investigated in two-stage culture in flasks, and carbon catabolic repression (CCR) was responsible for the regulation. The initial CS activities were enhanced after adding glucose to the medium, but stopped increasing afterwards. It suggested that glucose has positive effect on CS production. There were two reasons attributing to the decline of CS activities later stage:side-product(s) of glucose had harmful effect; on the other hand, ammonium released from the consumption of DL-ATC was used as nitrogen source, in that case, the consumption of DL-ATC was replaced. By calculating mass balance of nitrogen, some nitrogen substances were produced in the later stage, but negative effect on CS activities was not observed. Two-stage cultivation in5-L bioreactor showed CS activities decreased when dissolve oxygen stopped decreasing, probably because CS production was regulated by aerobic metabolism.Two-stage culture was performed in5-L bioreactor using glucose or acetic acid, respectively, for cell growth. The CS activities reach its maximum of302U/mL and283U/mL, and the yields on DL-ATC were18875U/g and21439U/g, respectively. CS activities decreased although cell density was enhanced from9.70g/L (glucose) to16.3g/L (acetic acid). CS activities from cells grew on glucose or acetic acid in two-stage culture in flasks showed different result, and it was much higher than that grew in acetic acid. It probably due to the metabolic pathway differences between glucose and acetic acid as carbon source. Some negative regulator belonging to Pseudomonas sp. F12exert effects. The most significant difference of growing in different carbon sources occurred among the central intermediary metabolic genes. The number of down-regulated genes was much higher than up-regulated genes in many functional groups in acetic acid growing cells.Studies on enzyme reactions revealed that hydrogen sulfide inhibited the degradation of L-cysteine. Thin layer chromatography determined that S-carbamyl-L-cysteine (SCC) was the intermediate. SCC can be hydrolyzed spontaneously or under SCC amidohydrolase, and L-cysteine was produced maily by hydrolysis under SCC amidohydrolase. It also showed that SCC amidhydrolase was much more stable than L-ATC hydrolase, which attribute to the low yield of L-cysteine.